PROJECT SUMMARY ? Project 2 Mutations in the parkin gene play a prominent role in Parkinson?s disease (PD) as mutations in parkin are the main genetic cause of autosomal recessive PD. Converging evidence suggests that inactivation of parkin through dopaminergic, oxidative and nitrosative stress as well as c-Abl phosphorylation may contribute to the degenerative process of sporadic PD. Parkin plays a pivotal role in the ubiquitin proteasomal pathway (UPP) by functioning as ubiquitin E3 ligase. Most disease causing mutations of parkin are thought to be loss of function mutations that ultimately lead to the absence of ubiquitination and the subsequent failure of UPP- mediated degradation of parkin substrates. Thus, the abnormal accumulation of parkin substrates could play a role in the demise of substantia nigra (dopamine) DA neurons in patients with parkin mutations, as well as, in patients with sporadic PD. Data acquired during the last funding period indicates that that accumulation of PARIS contributes to the loss of DA neurons in animal models of parkin or PINK1 inactivation, as well as models, of pathologic ?-synuclein (?-syn) induced neurodegeneration and loss of DA neurons. PARIS is a member of the KRAB-zinc finger protein (ZFP) family of transcription factors that contain a Kruppel-associated box or KRAB domain and four C2H2 zinc-fingers, which primarily function as transcriptional repressors. The mechanism of how PARIS represses the transcription of target genes and how elevation in PARIS levels leads to selective loss of DA neurons is not known. The study and characterization of how PARIS represses gene transcription and induces the selective loss of DA neurons due to parkin inactivation is the subject of this project. In Specific Aim 1 we will identify and characterize the regulatory mechanisms of the PARIS interactome. In addition, we will investigate the role of c-Abl phosphorylation on PARIS activity. In Specific Aim 2 we will characterize the genomic landscape of DA neurons and investigate the role of PARIS mediated site- specific methylation of gene(s) that are important in the survival of DA neurons in adult condition parkin knock out mice and in the gut-brain ?-syn PFF model of PD. Moreover, we will evaluate the levels of tyrosine phosphorylated parkin and PARIS in relation to methylation signatures in PD and PD related diseases. Based on our hypothesis that c-Abl may contribute to the pathogenesis of sporadic PD via tyrosine phosphorylation of parkin and PARIS Y137, we will assess the tyrosine phosphorylation of parkin and PARIS in serum derived L1CAM+ exosomes from the gut-brain ?-syn PFF model and the relationship of these serum L1CAM+ c-Abl marks will be correlated with the neuropathology and behavior of the gut-brain ?-syn PFF model. Finally, in Specific Aim 3 we will characterization of the role of PARIS mediated down regulation of Lmx1b via methylation of the Lmx1b promoter in mediating the loss of DA neurons in models of PD. Strategies will be employed to prevent the down regulation of Lmx1b that ultimately may identify new therapeutic strategies to treat PD.